title: Preparation and application of polyclonal antibodiesagainst KSHV v-cyclin.
abstract: We prepared rabbit polyclonal antibodies against Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded v-cyclin (ORF 72) and detected the natural viral protein using these polyclonal antibodies. Three antigenic polypeptides of v-cyclin were designed and synthesized. A fragment of the v-cyclin gene was cloned into a eukaryotic expression vector pEF-MCS-Flag-IRES/Puro to construct a recombinant vector, pEF v-cyclin. Then, pEF v-cyclin was transfected into 293T and EA.hy926 cells to obtain v-cyclin-Flag fusion proteins. Six New Zealand white rabbits were immunized with KLH-conjugated peptides to generate polyclonal antibodies against v-cyclin. The polyclonal antibodies were then characterized by ELISA and Western blotting assays. Finally, the polyclonal antibodies against v-cyclin were used to detect natural viral protein expressed in BCBL-1, BC-3, and JSC-1 cells. The results showed that using the Flag antibody, v-cyclin-Flag fusion protein was detected in 293T and EA.hy926 cells transfected with pEF-v-cyclin. Furthermore, ELISA showed that the titer of the induced polyclonal rabbit anti-v-cyclin antibodies was higher than 1:8,000. In Western blotting assays, the antibodies reacted specifically with the v-cyclin-Flag fusion protein as well as the natural viral protein. The recombinant expression vector pEF-v-cyclin was constructed successfully, and the polyclonal antibodies prepared can be used for various biological tests including ELISA and Western blotting assays.
authors.affiliation: Department of Microbiology and Immunology, Nanjing Medical University, Nanjing 210029, P. R. China; ; Department of Physiology, Xuzhou Medical College, Xuzhou, Jiangsu, 221000, P. R. China; Jiangsu 223300, China.
authors.forename: Min
authors.initials: M
authors.lastname: Xue
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Guo
authors.forename: Qin
authors.initials: Q
authors.lastname: Yan
authors.forename: Di
authors.initials: D
authors.lastname: Qin
authors.forename: Chun
authors.initials: C
authors.lastname: Lu
keywords: Kaposi's sarcoma-associated herpesvirus; polyclonal antibody; synthesized peptides; v-cyclin